The normal development of the vertebrate limb is dependent on the establishment of appropriate interactions among various cell and tissue types. One of the major components of the limb is skeletal muscle. The "blueprint" or plan specifying the precise arrangement and differentiation of the fiber types of skeletal muscle is established early in development and in close temporal association with innervation of the limb and the organizing influence of the connective tissue. The studies proposed here focus on the role of that these interactions play in establishing and maintaining the early pattern of muscle fiber types and controlling the subsequent development of the muscle fibers within the context of this "blueprint". These questions will be addressed in both the developing chicken and rat hindlimbs using a combination of embryo manipulations, in vitro culture procedures, and immunochemical analyses. In vivo, the expression of fiber type- and developmental stage-specific isoforms will be analyzed in limbs in which the normal interactions between the connective tissue and muscle and nerve and muscle has been altered or disrupted. In culture, the development and self-specializing capacity of muscle cells in a) the complete absence of interaction with other cell types, and b) in coculture with motorneurons and muscle "fibroblasts" will be examined. Preliminary data suggest that such interactions may affect expression by making explicit differences which are not normally apparent in culture. The identification of cell interactions that regulate and control the expression of muscle cell types and the reconstruction of these interactions in culture would be both an important conceptual and methodological advance in understanding the cellular mechanisms controlling gene expression and muscle fiber diversification during development.